The present invention relates to a chromosomal DNA sequence which codes for human lymphotoxin, a lymphotoxin expression vector, a lymphotoxin resistant cell line, a lymphotoxin resistant cell line transformed with the lymphotoxin expression vector, and a process for preparing lymphotoxin by employing the lymphotoxin resistant cell line or the lymphotoxin resistant mutant cell line transformed with the lymphotoxin expression vector.
Lymphotoxin (hereinafter referred to as "LT") has a toxicity selective to cancer cell and capable of leading the cancer cell to necrosis [Evans, C. H. et al. (1977), Cancer Res., Vol. 37, P898]. LT, which is expected for application as the antitumor agent, is a kind of lymphokine induced by stimulating lymphocyte from animal such as man or mouse with lectins such as phytohemagglutinin and concanavalin A or with phorbol ester [Devlin, J. J. (1984), Lymphokines, Vol. 9, P313]. As the representative LT producing cell in man, T cell which is selected by rosette formation with sheep red cell or B cell RPMI 1788 is known [Aggarwal, B. B. et al. (1984), J. Biol. Chem., Vol. 259, P686]. LT is a kind of glycoprotein [Toth, M. K. and Granger, G. A. (1979), Mol. Immunol., Vol. 16, P671] and has several kinds of molecular forms. LT has been studied from protein chemical aspects by several research groups. It has been reported that LT is constructed from a minimum unit having a molecular weight of about 20000, which associates with each other or forms a complex with other components [Aggarwal, B. B. et al. (1984), J. Biol. Chem., Vol. 259, P686].
It is known that LT is produced by the lymphocyte stimulated with phorbol ester, mitogen or the like. However, in this process, only extremely small quantities of LT is produced and a large amount of fresh lymphocyte is required, which make the process unsuitable for a large-scale production of LT. It is also known that LT is produced inductively by stimulating the established cell derived from lymphocyte (established cell line) with mitogen or the like. However, productivity of LT in this process varies depending on the ability of the cell employed and thus the process is not suited for a large-scale production. In recent years, cDNA of LT has been cloned and it has become possible to produce LT-like protein in E. coli [Gray, P. W. et al. (1984), Nature, Vol. 312, P721]. However, in most cases, LT-like protein produced in microorganism has a N-terminal different from that of the natural one, since the microorganism has a protein synthetic mechanism which is somewhat different from that of the animal cell. Further, LT-like protein produced in a microorganism does not contain a sugar moiety linked to the protein whereas natural LT does.
A structure of LT sugar moiety remains mostly unknown. It is not known whether there is a difference in structure and antigenicity between LT produced by the human cell and LT produced by the non-human cell. However, LT produced by a human cell is supposed to closely resemble the natural LT and to have more safety than LT produced by the non-human cell. When LT is produced by the non-human cell, there is a possibility of contamination of LT product with constructive materials such as proteins or secretions from cells other than human cells and thus one can expect problems such as allergic reaction or shock when the LT product is administered in a long term as a therapeutic agent. On the other hand, LT product produced by the human cell contains only human material and does not contain any material other than that present in human blood, which improves the safety of the product.
From the above point of view, a process for preparing natural LT having the sugar moiety have been investigated.
In recombinant DNA technique, various problems are raised.
It is known that many proteins in higher organisms are coded on the chromosomal DNA sequence in several separated portions DNA sequence which codes matured mRNA is referred to as the "exon" while the separating sequence is referred to as the "intervening sequence" or "intron". Although the biological roles or functions of the introns remains still almost unknown, it is known that a gene without the intron such as those coding ovalbumin [Wickens, M. P. et al. (1980), Nature, Vol. 285, P628] or viral protein [Lai, C-J. et al. (1979), Proc. Natl. Acad. Sci. USA, Vol. 76, P71] produces far less protein in the animal cell as compared with the intron containing gene. It is also known that accumulation of stable mRNA occurs when the intron from .beta.-globin gene is added to SV40 gene devoid of the intron [Hamer, D. H. et al. (1979), Cell, Vol. 18, P1299].
Removal of the sequence corresponding to the intron from nascent mRNA, which is transcribed from gene, is referred to as the "splicing". The splicing is presumed to be necessary for accumulation of stable mRNA or for transfer of mRNA from nucleus to cytoplasm.
For the expression of normal and functional protein, it is indispensable that the splicing occurs at the correct position. It has been reported that the abnormal splicing was observed when the insulin gene is linked with the promoter region of SV40, which is then introduced into COS cell [Laub, 0. et al. (1983), J. Biol. Chem., Vol. 258, P6043]. It is also known that the expression of amylase gene is conducted through tissue specific splicing, wherein salivary gland amylase and liver amylase are synthesized by way of two different splicing process from one and the same gene [Young, R. A. et al. (1981), Cell, Vol. 23, P451]. Also in SV 40 [Berk, A. J. et al. (1978), Proc. Natl Acad Sci. USA, Vol. 75, P1274], adenovirus [Chow, L. T. (1977), Cell, Vol. 12, Pl] and the like, more than one mRNA and protein are synthesized through different splicings from one and the same gene.
Therefore, the correct splicing is required in order to produce LT by introducing LT gene with the intron into the cultured animal cell. The present inventors have found that normal splicing occurs to secrete LT into the culture medium when LT coding chromosomal DNA sequence is linked with DNA sequence of the promoter region which functions in the cultured animal cell and initiates mRNA synthesis, which is then introduced into the various cultured animal cells.
For the production of LT which has the same amino terminal as that of natural LT and has the sugar moiety, gene recombination technique can be applied where the cultured animal cell is employed as the host. In this case, the introduction of only LT gene into the cultured animal cell is presumed not to lead to LT production. That is, LT is an inducible protein and the expression of LT gene is suppressed at the genetic level. In fact, the present inventors found that the amount of produced LT was quite small even if LT gene, which included the expression controlling region such as promoter, was introduced into various cultured cells. This suggests that some improvement must be made on LT gene in order that the cell wherein LT gene is introduced has the effective productivity of LT. The abovementioned improvement on LT gene is one of the main purposes of the present invention.
In 1981, Banerji et al. showed that the expression of rabbit .beta.-globin gene is enhanced by the 72 bp repeat present in the vicinity of replication origin of SV40 DNA closely linked to the .beta.-globin gene [Benerji, J. et al. (1981), Cell, Vol. 27, P299]. The enhancing effect of the 72 bp repeat in SV40 was observed at every position and direction of the linked sequence. Such relatively short DNA sequence which enhances the gene expression is referred to as the enhancer sequence. It was found that the enhancer sequence is also present in the genom of Rous sarcoma virus (RSV), polyoma virus, bovine papilloma virus and the like [Gluzman, Y. and Shenk, T. ed., Current communications in molecular biology, Cold Spring Harbor Laboratory (1983)]. It is also known that the enhancer is present in the intron of immunoglobulin gene [ibid].
The expression vector of LT utilizing such enhancer sequence is characterized by that it reduces the possibility of abnormal splicing and of the production of abnormal protein since the original promoter of LT gene functions.
On the other hand, it appeared difficult to obtain the subculturable cell line transformed with the LT expression vector since LT is strongly toxic to the cell and thus the cell transformed with the LT expression vector will be killed by the self-produced LT. However, the present inventors have found that the transformed BHK cell, wherein the LT expression vector is introduced, can produce LT, the transformed BHK cell being subculturable to produce LT for a long period of time. The present inventors have tested the susceptibiliy of BHK cell to LT and have found that the BHK cell is resistant to LT. On the other hand, the cells such as some CHO line, FL and WISH were sensitive to LT and died. When these LT-sensitive cells were transformed with the LT expression vector, the resultant transformed cells showed no production of LT or production of only a very small amount of LT. Vero and WI-26 VA4 cells, which are resistant to LT like BHK cell, gave the transformed cell line which stably produced LT.
Hitherto, a large number of the cell has been established. Among them, some established cell line while showing sensitivity to LT has excellent characteristics for cell breeding such as high efficiency of transformation, facility in obtaining the transformed cell line, low serum-dependency in cell growth, rapid growth, facility in a large-scale culture, high ability of synthesizing or secreting the protein having well-known properties of the cell and having specific genetic property. Therefore, also in these established cells, it is very important for the production of LT to establish the technique to obtain the transformed cell line which stably produces a large amount of LT.
The present inventors have established the technique to separate the LT resistant cell, from which the transformed cell line being obtained to produce LT stably. These cells could produce LT also in the serum free medium. The employment of the serum free medium in the LT production not only faclitates recovery and purification of LT from the medium but also prevents contamination of the products with serum component.
When the gene is introduced into the cell, the gene is sometimes incorporated into the host chromosomal DNA stably. The incorporated position of the gene in the chromosome is apparently random and a copy number of the incorporated DNA is also irregular. When LT gene is introduced into the cell, the incorporated position and the copy number vary in every cell, each cell producing a different amount of LT. Therefore, the cells which produce various amount of LT can be obtained by cloning the cell. The amount of the produced LT is presumed to have correlation with the copy number of LT gene and the cell having the increased number of LT gene is expected to show the improved productivity of LT.